The purpose of this research is to improve the dietary treatment of hypercholesterolemia by understanding more fully how dietary factors interact with mechanisms of cholesterol absorption. Cholesterol absorption is a critical control point for cholesterol metabolism because it affects both dietary and endogenous recirculating biliary cholesterol. The central hypothesis of this proposal is that certain lipids (phytosterols, squalene and oxysterols), in the quantities found in western diets, have physiologically important effects on cholesterol absorption and metabolism. The aims involve human subjects, animals and cultured cells and rely heavily on stable isotopic tracers and newly developed mass spectroscopic methods of analysis. Dietary supplementation with phytosterols is known to reduce cholesterol absorption. During the current funding period we have demonstrated that the quantity of phytosterols contained in normal diets is sufficient to reduce cholesterol absorption. Here we propose to use a novel phytosterol-deficient baseline diet in controlled feeding studies to test the hypothesis that natural food phytosterols lower LDL cholesterol. We will also explore the interaction of phytosterols with ezetimibe. Squalene is known to be a cholesterol precursor but whether dietary squalene contributes significantly to cholesterol synthesis in man has not been adequately addressed. We will measure squalene absorption and quantify its effect on cholesterol biosynthesis and LDL cholesterol. Oxysterols are known to regulate cholesterol absorption by binding to LXR/RXR nuclear receptors in enterocytes; however the effect of dietary oxysterols on cholesterol absorption has not been addressed. We propose to measure the metabolism of oxysterols in humans and to define their effects on cholesterol absorption. Finally we will use cholesterol enantiomer, a new synthetic probe which is not absorbed but which has identical physical properties as cholesterol, to trace absorption pathways. Successful completion of this work will provide new directions for improving circulating lipoproteins through dietary counseling as well as by changes in industrial food manufacturing.